1. Field of the Invention
This invention concerns the determination of endotoxin in fluids using a Limulus amebocyte lysate based assay.
2. Description of Related Art
The coagulation phenomena of the pro-clotting enzyme of the amebocyte lysate from the blood of the horseshoe crab by bacterial endotoxin has been known and reported for many years, see, for example Levin, J. and Bangs, F. B., "Clottable Protein in Limulus: Its Localization and Kinetics of its Coagulation by Endotoxin," Thomb. Diath. Heamortz. 19, pg. 186 (1968). The clotting mechanism has been subject to in-depth studies such as the study by Nakamura, S. et al., "Amino Acid Sequence Studies on the Fragments Produced from Horseshoe Crab Coagulogen during Gel Formation: Homologies with Primate Fibrinopeptide B", Biochemical and Biophysical Research Communication, 72(3), p. 902 (1976).
The coagulation of Limulus amebocyte lysate, hereinafter LAL, involves the endotoxin activation of a pro-clotting enzyme in the presence of divalent cations, e.g. Ca.sup.++, Mg.sup.++, Sr.sup.++ or Mn.sup.++, with the resulting activated enzyme cleaving a clotting protein (coagulogen) at the C-carboxyls of contained glycine and arginine units. The cleaved units of the coagulogen remain attached by disulfide bridges and undergo polymerization to effect a clot. In addition to these known components of the amebocyte lysate, there are many other proteins and a known inhibitor of a lipoprotein nature. The mechanism of modulation of the coagulation reaction by the inhibitor and other proteins has not yet been determined.
Because of the coagulation characteristic of LAL in the presence of bacterial endotoxin (pyrogen), LAL formulations have become commercially important reagents for use in endotoxin assays for quality control in the manufacture of various fluids of pharmaceutical or medical interest which are normally administered parenterally. Such fluids include water for injection; water for irrigation; lipid emulsions for intravenous feeding; aqueous emulsions of vegetable oil; salt solutions, e.g. parenterally administered sodium chloride solutions including sodium chloride for injection USP, sodium chloride for irrigation USP, sodium chloride for inhalation, and lactated Ringer's solution; and blood derivatives, e.g., normal serum albumin, plasma protein fraction and anti-hemophilic factor USP, immune globulin, Rho(D) immune globulin and antihuman globulin serum.
The formulation of LAL reagents and the improvement in LAL procedures has progressed to the point that an LAL assay is the most sensitive and practical endotoxin test that is known. The LAL assay can detect, with the formation of a clot, as little as 10.sup.-12 grams/ml of endotoxin. A Health Industries Association Study [Dabah, et al., "HIMA Collaborative Study for the Pyrogenicity Evaluation of a Reference Endotoxin by the USP Rabbit Test", HIMA Document No. 7, vol. 1 (May, 1979)] showed that the United States Pharmacopeia (USP) rabbit pyrogen assay can detect approximately 10.sup.-9 grams/ml of endotoxin. Therefore, the LAL assay is approximately 100 times as sensitive as the USP rabbit pyrogen assay. In addition to its advantage of sensitivity, the LAL assay is simpler to perform and can be completed in approximately one hour as opposed to three hours for the rabbit assay.
The assay of endotoxin detected by clotting of LAL is essentially a kinetic assay. Endotoxin activates the clotting enzyme, the clotting enzyme cleaves the coagulogen, and the cleaved coagulation aggregates to form a gel. More endotoxin results in more rapid accumulation of cleaved coagulogen and in faster gel formation, and so the time required to form the gel is less when more endotoxin is present. In other words, the endotoxin concentration is inversely related to the gelling time in the assay.
There are difficulties associated with precise determination of clotting time, as required by this assay methodology. Aggregation, flocculation and clotting are successive stages in a complex physical phenomenon involving a multiplicity of physical forces and probably multiple components. The boundaries between the stages are not clearly marked, but rather they are subjectively established and can differ when established by different observers. LAL is a chemically complex mixture, and apparently small changes in composition can have profound effects on the course of the complex coagulation phenomenon. Finally, an assay based on the subjective determination of gel formation is inherently difficult to automate. An alternative assay methodology was developed to overcome some of the difficulties associated with accurately determining gel formation and the associated endotoxin concentration.
The use of chromogenic substrates has become a means to both study and clinically monitor various enzymes and inhibitors in the complex coagulation processes of man. An extensive list of enzyme specific substrates are commercially available for measuring enzymes such as trypsin, thrombin, thromboplastin, plasmin, Kalikrein, Urokinase, and plasminogen. These synthetic substrates provide the investigator with an important tool to monitor the hemostatic state of certain aspects of the coagulation process in vitro.
Iwanaga, et al., "Chromogenic Substrates for Horseshoe Crab Clotting Enzyme: Its application for the Assay of Bacterial Endotoxin", Hemostasis 7:183-188 (1978), report that synthetic substrates can be used to measure the level of endotoxin activated pro-clotting enzyme in LAL prepared from the blood of both the Japanese (Tachypleus tridentatus) and the American (Limulus polyphemus) horseshoe crabs. One advantage of chromogenic substrates in an LAL assay relative to a conventional LAL gelation test is that the amount of activated clotting enzyme can be quantified. The use of certain synthetic peptide-type substrates in LAL assay to measure bacterial endotoxins quantitatively has been described in U.S. Pat. No. 4,188,264. The disclosure of the patent teaches a peptide substrate with a structure consisting of L-amino acids in the sequence R.sub.1 -gly-arg-R.sub.2 where R.sub.1 represents an N-blocked amino acid and R.sub.2 is a group which can be released by enzymatic hydrolysis to yield a colored compound, HR.sub.2.
Another patent, U.S. Pat. No. 4,510,241, discloses an improved chromogenic peptide substrate for use in LAL-type assays. This improved substrate differs most significantly from the previous substrate in that the gly moiety is replaced in the sequence by ala or cys.
During an LAL-type assay using one of these substrates, the pro-clotting enzyme (a serine protease) in the LAL is activated by endotoxin and cleaves the peptide chain on the carboxyl side of arginine so as to release the chromogenic group and form a marker compound which can be easily read by means such as spectrophotometry.
A number of drawbacks to the LAL assay remain, despite numerous modifications designed to improve quantitation, sensitivity, speed, and precision. Foremost among these is the limited range of sensitivity (about 1 order of magnitude) with either the turbidimetric or the chromogenic fixed incubation time (endpoint method). A second drawback to endpoint methods is the requirement that an operator must end or read the reaction at a precise time. In the chromogenic test, acid must be added to stop the test. The optical density can be read in a spectrophotometer at any time thereafter. In the endpoint turbidimetric method, the reaction cannot be stopped without destroying the turbidity but must be read immediately after a specific incubation period.
An alternative approach to improving the LAL-type assay was to improve the precision of measuring the coagulation phenomenon. This was done by focusing on the increase in turbidity of the assay solution as aggregation proceeded, rather than waiting for gelation (Young, et al., (1972) J. Clin. Invest., 51:1790-1797). The kinetic turbidimetric LAL assay provides several potential improvements over endpoint methodology. The kinetic turbidimetric method uses a single reagent and does not require operator attention after initiation of the test, thus precision, speed, and accuracy are all improved. Most importantly, the range of the test could be increased from 1 to greater than 5 orders of magnitude. Unfortunately, turbidity determinations made with a spectrophotometer use the decrease in transmitted light caused by physical blocking, and thus Beer's law does not apply. Particle size and number, and reflected and refracted light all affect measurement to various degrees. Though elegant electronic filtering coupled with computer smoothing of data have been successfully developed and used for interpreting kinetic turbidity results (Novitsky, et al., p. 189-196, and Remillard, et al., pp. 197-210, both in Watson, et al., "Detection of Bacterial Endotoxins with the Limulus Amebocyte Lysate Test," Alan R. Liss, Inc., New York, 1987), these methods are available only on specialized optical readers. In addition, some products must be diluted extensively to be assayed at all with the kinetic turbidimetric method.